UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Fermentation of sulfite spent liquor Nishikawa, Masabumi 1968

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1968_A7 N48.pdf [ 3.61MB ]
Metadata
JSON: 831-1.0059117.json
JSON-LD: 831-1.0059117-ld.json
RDF/XML (Pretty): 831-1.0059117-rdf.xml
RDF/JSON: 831-1.0059117-rdf.json
Turtle: 831-1.0059117-turtle.txt
N-Triples: 831-1.0059117-rdf-ntriples.txt
Original Record: 831-1.0059117-source.json
Full Text
831-1.0059117-fulltext.txt
Citation
831-1.0059117.ris

Full Text

FERMENTATION OF SULFITE SPENT LIQUOR by MASABUMI NISHIKAWA B.Eng. Kyoto University, JAPAN, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF M.A.Sc. i n the Department of Chemical Engineering We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1968 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n -t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t n f T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8 , C a n a d a D a t e i Abstract Fermentation of s u l f i t e spent l i q u o r with Propionibacterium f r e u d e n r e i c h i i was done to produce v o l a t i l e acids (acetic and pro-pionic) and Vitamin B i 2 - It was found that i n addition to producing these compounds some reduction i n the p o l l u t i o n p o t e n t i a l (COD) of t h i s waste product was achieved. Better growth resulted i f the spent l i q u o r was f i r s t treated to remove l i g n i n and calcium compounds. An e x i s t i n g spectrophotometry assay technique f o r measuring Vitamin B12 content was modified for use i n the presence of s u l f i t e spent l i q u o r . i i TABLE OF CONTENTS ABSTRACT i LIST OF TABLES i v LIST OF FIGURES v 1. INTRODUCTION 1 1-1 Metabolism 4 1-2 Vitamin B 1 2 7 1-3 Propionibacterium f r e u d e n r e i c h i i 9 1- 4 Objective of t h i s research 12 2. EXPERIMENTAL TECHNIQUE 12 2- 1 Analysis of wood sugars 12 2-2 V o l a t i l e acid analysis 12 2-3 Lignin analysis 13 2-4 Chemical oxygen demand 13 2-5 Measurement of b a c t e r i a l growth 13 2-6 VB 1 2 analysis 14 2-7 S u l f i t e spent l i q u o r preparation 21 2-8 Synthetic media preparation 25 2-9 Fermentation technique 26 2-9-1 S t e r i l i z a t i o n 26 2-9-2 Test tube growth test 26 2-9-3 Bellco glass fermentor 27 2- 9-4 11 fermentor 27 3. RESULTS 27 3- 1 Spectrophotometry analysis 27 i i i 3-2 Comparison of photometric and microbiol o g i c a l assay 27 3-3 Test tube growth te s t s 31 3-4 Bellco glass fermentor and 7£ fermentor 31 3-4-1 COD 45 3-4-2 V o l a t i l e acids 45 3-4-3 VB 1 2 45 4. DISCUSSION 48 5. CONCLUSIONS 57 6. RECOMMENDATIONS 57 ACKNOWLEDGEMENT 60 REFERENCES 61 APPENDIX 67 1-1 Sugar analysis 67 1-2 V o l a t i l e acids analysis 68 1-3 Ratio of propionic a c i d to a c e t i c acid 68 1-4 Lignin measurement 69 1-5 Tu r b i d i t y 70 2 COD 70 iv LIST OF TABLES 1. Cobamides 6 2. V B 1 2 y i e l d of previous workers 11 3. T y p i c a l analysis of SSL from Columbia Cellulose and of a spruce SSL 23 4. T e s t r o f accuracy of the spectrophotometric method for V B i 2 29 5. Comparison of microbiological and spectrophotometric assay 32 6. Test tube growth test i n non-treated spent l i q u o r 34 7. Test tube growth te s t i n treated spent l i q u o r 35 8. Bellco glass fermentor fermentation 37 9. Bellco glass fermentor fermentation 38 10. . Be l l c o glass fermentor fermentation 39 11. 11 fermentor fermentation 41 12. Y i e l d of v o l a t i l e acids 44 13. V B 1 2 a c t i v i t y d i s t r i b u t i o n i n 11 fermentation 47 V LIST OF FIGURES 1. Acids metabolic pathway 5 2. Cyanoeobalamin s t r u c t u r a l formula 8 3. P. f r e u d e n r e i c h i i i n SSL media 10 4. Spectra of VB 1 2 17 5. Spectra of VB 1 2 i n SSL 18 6. Spectra of SSL 19 7. M i c r o b i o l o g i c a l assay standard curve 22 8. S u g a r - b i s u l f i t e complex i n SSL 24 9. Diagram of 71 fermentor 28 10. Test of spectrophotometric assay 30 11. Comparison of spectrophotometric and m i c r o b i o l o g i c a l assay 33 12. Growth curve of the t e s t tube growth t e s t 36 13. 500 ml Bellco fermentor i n SSL medium 40 14. 7£ fermentor fermentation i n synthetic medium 42 15. 7& fermentor fermentation i n SSL medium 43 16. COD vs. sugar content 46 17. Volume base fermentation rate i n 7£ fermentor 51 (synthetic medium) 18. Volume base fermentation rate i n Bellco glass fermentor 52 19. Volume base fermentation rate i n 7% fermentor (SSL medium) 53 20. Proposed flow sheet 58 1 1 . I n t r o d u c t i o n The d i scharge o f waste l i q u o r from i n d u s t r i a l opera t ions can l e a d t o a major water p o l l u t i o n prob lem. Th is i s p a r t i c u l a r l y t r u e o f the e f f l u e n t d ischargedf rom s u l f i t e p u l p i n g o p e r a t i o n s . The p o l l u t i o n caused by t h i s s u l f i t e spent l i q u o r (SSL) r e s u l t s from suspended s o l i d s , d i s s o l v e d o r g a n i c subs tances , and chemicals which are t o x i c to the i n h a b i t a n t s o f the r e c e i v i n g water and perhaps even t o the users o f t h i s water ( 1 - 8 ) . In the s u l f i t e process about 8 tons o f spent l i q u o r are d i s -charged per ton o f p u l p produced. In t h i s amount o f spent l i q u o r are found 1000 l b s o f l i g n i n complexes, 500 lbs o f wood sugars ( reducing substances) and about 20 l b s o f suspended s o l i d s (9)• Suspended s o l i d s may be removed by s c r e e n i n g , f i l t r a t i o n , s e t t l i n g , or f l o t a t i o n ( 1 ) . Th is suspended m a t t e r , which i s most l y f i b r e o r b a r k , causes i r r i t a t i o n o f the g i l l t i s s u e o f f i s h . A l s o as c e l l u l o s e i s r e l a t i v e l y r e s i s t a n t t o decay , t h i s f i b r e d e b r i s tends to b l a n k e t the bottom of the r e c e i v i n g stream c r e a t i n g anaerob ic c o n d i t i o n s on the bottom. Th is smothers the l i f e at the stream bottom and may produce unpleasant s m e l l i n g gases . Where r e g u l a t i o n s are i n f o r c e a s t i p u l a t e d maximum ± s 0 . 3 l b s o f suspended s o l i d per U.S. g a l l o n d i s -charged (10) . The average i n d u s t r y d i s c h a r g e i s c o n s i d e r a b l y h i g h e r . Tox i c m a t e r i a l s such as r e s i n a c i d soaps , mercaptans , e t c . are u s u a l l y n e u t r a l i z e d by pH adjustment . However not much i s r e a l l y known about t h i s . L i g n i n complexes and t a n n i n s cause water d i s c o l o r a t i o n as w e l l as foaming when d i s c h a r g e d from the m i l l . Such c o n d i t i o n s a r e , i f n o t h i n g 2 else, unsightly. Foaming tends to reduce the eff i c i e n c y of most solids removal techniques. Dissolved, oxidizable chemicals such as wood sugars, l i g n i n compounds, etc. are naturally oxidized by microbes i n the water. This process removes oxygen from the receiving stream; 400 lbs or more of oxygen may be required to oxidize the effluent discharged i n the pro-duction of 1 ton of s u l f i t e pulp. Since water saturated with oxygen contains only 8-11 ppm of oxygen, oxygen depletion occurs i n the water rather rapidly unless oxygen can be dissolved i n the water at a rate equal to or greater than the rate at which i t i s being used up. I f this cannot be done the aquatic fauna suffocate from lack of oxygen. The solution to these p o l l u t i o n problems may be i n finding a use for the spent liquor, or a treatment which e f f e c t i v e l y neutralizes i t . It i s possible that the s u l f i t e process w i l l be displaced by the kraft process and thus the problem w i l l no longer e x i s t , as the water p o l l u t i o n load from a kraft m i l l i s much less than that of a s u l f i t e m i l l . However, the s u l f i t e process i s s t i l l with us and i t may well be that the k r a f t process w i l l be leg i s l a t e d out of existence unless i t can solve i t s a i r p o l l u t i o n problems. Thus there i s some j u s t i f i c a t i o n for studying the u t i l i z a t i o n of s u l f i t e spent liquors. Some uses for the liquor have been proposed (11-19); these are i n road binding, adhe-sives, and as a f u e l . These are low cost products and require concentra-ti o n of the liquor v i a a costly evaporation process. Small quantities of l i g n i n i n spent liquor are used i n the manufacture of v a n i l l i n (20, 21, 22), however presently operating plants have s u f f i c i e n t capacity to meet world demands. Some of the wood sugar i n the s u l f i t e spent 3 l i q u o r i s pentose from which f u r f u r a l can be manufactured by acid d i s t i l l a t i o n (23) or by an u l t r a s o n i c technique (24) but neither process has proven a t t r a c t i v e economically. Fermentation of the wood sugars has been suggested. A number of m i l l s are i n existence pro-ducing ethanol and yeast by fermentation of the sugars i n the s u l f i t e spent l i q u o r (25, 26, 27). Since considerable amounts of oxygen are required to oxidize these sugars, t h e i r removal might e f f e c t i v e l y lower the p o l l u t i o n load of the e f f l u e n t . However i t should be noted that i n Canada, U.S.A., U.S.S.R. and Japan l i t t l e recovery i n t h i s form i s pra c t i c e d . Let's look at fermentation i n more d e t a i l . Stream p o l l u t i o n must be reduced. This requirement w i l l r e s u l t from l e g i s l a t i o n , or enlightened management att i t u d e s . I f i t must be reduced any return on the investment required to reduce i t w i l l be de s i r a b l e . The more valuable the product from a spent l i q u o r treatment i s , the greater i s the chance f or producing an acceptable return on investment. Jensen (28) and Van N i e l (29) suggested the use of prop i o n i b a c t e r i a f o r the production of organic acids by fermentation of wastes containing sugars. A valuable byproduct from such a fermentation would be vitamin (y^iz) (30, 31, 32, 33). The production of acetic acid and propionic acid from s u l f i t e spent l i q u o r (Ca base) by fermentation with p r o p i o n i b a c t e r i a has been reported (34, 35, 36, 37). It i s known that some species of propioni-b a c t e r i a produce V B 1 2 i f cobalt ion i s present i n low concentration (38, 39, 40, 41). Some microorganisms make V B ^ - l i k e compounds which have l i t t l e or no b i o l o g i c a l a c t i v i t y but i t has been shown that 4 Propionibacterium f r e u d e n r e i c h i i produces active 5,6-dimethyl benzimidazole hydroxo cobalamin ( O H B 1 2 ) (42) insi d e i t s c e l l body (43). Some cobamides found i n microorganisms are shown i n Table 1. The V B 1 2 y i e l d s recorded by previous workers from the fermentation of materials other than pulp m i l l wastes are presented i n Table 2. The present work considers the production of V B 1 2 and organic acids from calcium base s u l f i t e spent l i q u o r by fermentation with Propionibacterium f r e u d e n r e i c h i i (ATCC 6207). 1-1 Metabolism Propionibacteria can use both hexose and pentose for growth. The metabolic pathway for the formation of organic acids has been studied by many workers (44-54). However the complete system of reactions which occur i n s i d e the c e l l i s not yet f u l l y understood. According to Phares and Delwiche (50) Propionibacterium shermanii was able to e f f e c t a quantitative conversion of s u c c i n i c acid to propionic acid and C0 2 under anaerobic conditions. Smith (51), Swick (52), F l a v i n , et a l (45-49), and Staatman, et a l (55) believe that methyl malonyl CoA, s u c c i n y l CoA, and propionyl CoA are intermediates i n propionic acid metabolism. Methyl malonyl CoA i s derived from pyruvic acid which i s the end product of sugar metabolism v i a g l y c o l y s i s . A suggested meta-b o l i c route to propionic acid and acetic ac i d i s shown i n F i g . 1. DBCC (5,6-dimethyl benzimidazolyl cobamide CoA) which i s a co-factor i n transmethylation reactions (Fig. 1) i s derived from V B 1 2 (43, 56, 57, 58). However d e t a i l s of the conversion of V B 1 2 to DBCC are not yet known. 5 Table 1 Cobamides Cobamide Sources Reference A c t i v i t y (111, 113)* L. leichmanii Ochromonas Hydroxo Cobalamin P. f r e u d e n r e i c h i i Streptmyces species Perlman (42) Pseudo VB 1 2 (Adenine hydroxo cobalamin) P. arabinose P. pentosaceum P. shermanii Anaerobes Perlman (42) Hodgkin (59) P f i f f n e r (96) 40 - 75 0.03 Factor A (2-Methyl-a adenyl cobamide) P. f r e u d e n r e i c h i i P. th o e n i i P. pentosaceum Porter (97) 1! II 17 - 40 s l i g h t Factor B (cobinamide) C a l f rumen Nocardia rugosa P. shermanii Ford (98) Di Marco (99) Pawelkiewitz (100) 0 0.03 Factor C (a-guanyl cobamide) N. rugosa E. c o l i P. shermanii B a r c h i e l l i (101) Ford (102) Pawelkiewitz (100) 10? 0 Ochromonas and L. leichmanii indicate the microbiological method used to measure the V B i 2 a c t i v i t y . A c t i v i t y i s 100 for cyanocobalamin. 7 1-2 Vitamin B 1 2 VB^2 was first isolated from liver concentrate (40). Rickes et al (41) described a microbial synthesis using streptmyces griseus. Later many other microorganisms were discovered that could synthesize V B i 2 - VB;L2 1 S essential for normal blood formation and neural function in mammals as well as playing a role in several other mammallian growth processes. VBi 2 is a water soluble vitamin known chemically as a cobalamin. It is a complex co-ordinating compound containing a tri-valent cobalt ion with a co-ordination number of six. The complete structural formula for cyanocobalamin (CNBi2) which was reported by Hodgkin, et al (59) is given in Fig. 2. Cyanocobalamin is a neutral molecule; the cyano group attached to the cobalt atom can be replaced by other ions or groups to yield other cobalamins such as hydroxocobalamin, chloro-cobalamin, nitro-cobalamin, or thiocyanocobalamin. Of these hydroxo-cobalamin is reported to be the most biologically active cobalamin (43). Al l these cobalamins can be converted to the stable cyanocobalamin by treatment with cyanide ion. VBi 2 is now obtained almost exclusively from microbial sources, either as a primary fermentation product, or as a byproduct in the production of certain antibiotics. The importance of microbial pro-duction of VBi 2 was summarized by Smith (60) as follows: "It seems probable that the only primary source of V B 1 2 in nature is the metabolic activity of microorganisms; there is no con-vincing evidence for its elaboration in tissues of higher plants or animals. It is apparently synthesized by a wide range of bacteria and actinomycetes though apparently not to any extent by yeast or fungi." 8 9 Highly p u r i f i e d V B i 2 i s necessary for the treatment of pernicious anemia and n u t r i t i o n a l d e f i c i e n c i e s i n humans, but at the present time the most promising, large scale market for V B i 2 appears to be as a supplement i n animal feeds. 1-3 Propionibacterium f r e u d e n r e i c h i i This b a c t e r i a was found i n dairy products and i s a gram-p o s i t i v e , non-spore forming, non-motile b a c t e r i a . It grows under both aerobic and anaerobic conditions u t i l i z i n g carbohydrates, polyal.co.hbls, or l a c t i c acid to produce propionic acid, a c e t i c a c i d , and C0 2 (53). Eleven species of p r o p i o n i b a c t e r i a have been described (29, 54, 61). On yeast extract-glucose medium P. f r e u d e n r e i c h i i and P. shermanii grow as small c o c c i . P. f r e u d e n r e i c h i i i s rod shaped i n the synthetic medium (1% Beef extract, 0.3% yeast extract, 1% peptone, 0.5% dextrose, 0.1% soluble starch, 0.05% cysteine hydrochloride, 0.5% sodium c h l o r i d e , 0.3% sodium acetate and 0.05% agar) but changes i t s shape to c o c c i i n the s u l f i t e spent l i q u o r medium. A photograph of P. f r e u d e n r e i c h i i i n SSL medium i s shown i n F i g . 3. Perlman, et a l (42) found that P. f r e u d e n r e i c h i i produces hydroxocobalamin which i s converted to DBCC much f a s t e r than cyano-cobalamin or other cobalamins (43). T y p i c a l production figures for the synthesis of V B i 2 by t h i s b a c t e r i a are provided i n Table 2. The optimal temperature for the synthesis of V B i 2 by P. freu-d e n r e i c h i i i s about 30°C and the optimal pH i s 6.5 to 7.0 (62). It has been experimentally demonstrated that a good y i e l d of V B i 2 can not be expected under s t r i c t l y aerobic or s t r i c t l y anaerobic conditions ( 3 0 , 62, 63). 10 x 9000 Table 2 VB12 Y i e l d of Previous Workers Bacterium Y i e l d (mg/1) Reference P. f r e u d e n r e i c h i i 3.0 2.4 3.0 0.4(24hrs) 3.7 Leviton Aso Sudarsky Perlman Riley (30) (33) (65) (42) (64) P. shermanii 3.0 Hinz (103) Streptmyces species 5.7 Pagona (104) S. griseus 0.3 Wood Dulaney (no) (105) S. olivaceus 3.3 3.0 1.7 H a l l Hester P f e i f e r (106) (107) (63) S. fradiae 0.7 Nelson (108) B a c i l l u s megatherium 0.45 0.48 Lewis Ga r i b a l d i (109) (62) Aerobacter aerogenes 0.42 Hodge (32) Flavobacterium solare 0.6 Petty (76) 12 Ri l e y , et a l (64) reported that factor B (VB 1 2 less the nucleotide; cobinamide) was formed under anaerobic conditions and that a nucleo-t i d e was introduced i n t o factor B under aerobic conditions to give VBi2- Thus f or a good y i e l d of V B j 2 they suggested that the fermen-t a t i o n be conducted i n two stages, the f i r s t anaerobically to produce factor B and the second a e r o b i c a l l y to complete conversion to V B i 2 . Aso, et a l (33) noted that a g i t a t i o n was necessary f o r a good y i e l d of product and also that fermentation with a g i t a t i o n but without aeration gave a good y i e l d of V B i 2 . A g i t a t i o n e f f e c t s on V B i 2 production have also been discussed by P f e i f e r (63), G a r i b a l d i (61), and Sudarsky(64). 1- 4 Objectives of t h i s research The goals of the present work are 1) to get some quantitative data on the production of VB 1 2 and organic acids by fermentation of s u l f i t e spent l i q u o r with P. f r e u d e n r e i c h i i and 2) to determine i f such a fermentation can e f f e c t i v e l y reduce the p o l l u t i o n load of s u l f i t e spent l i q u o r by removing some or a l l of the wood sugars and l i g n i n s . 2. Experimental technique 2- 1 Analysis of wood sugars The t o t a l reducing substances i n s u l f i t e spent l i q u o r was measured by the method of Sieber (66). This i s also Swedish standard method CCA 11 (1941). For further d e t a i l s see appendix 1. 2-2 V o l a t i l e a c i d analysis The t o t a l v o l a t i l e acids (propionic acid and ac e t i c acid) i n 13 the fermentation broth were measured by a d i s t i l l a t i o n method and checked by steam d i s t i l l a t i o n . The r a t i o of propionic acid to a c e t i c acid was measured by steam d i s t i l l a t i o n (67). See appendix 1 for d e t a i l s . 2-3 Lignin analysis Analysis for l i g n i n i n the s u l f i t e spent l i q u o r followed the method of Partansky and Benson (68). D e t a i l s are given i n appendix 1. 2-4 Chemical oxygen demand (C.O.D.) This was determined by ASTM method D 1252-58T (68). The COD measurement i s described i n appendix 2. 2-5 Measurement of b a c t e r i a l growth B a c t e r i a l growth was followed by measuring the t u r b i d i t y of a suspension of c e l l s produced as follows. A sample of fermentation broth was centrifuged for 15 minutes at 2500 g's to remove the b a c t e r i a plus some p r e c i p i t a t e d s o l i d s from the spent l i q u o r . These s o l i d s and b a c t e r i a were resuspended i n d i s t i l l e d water and centrifuged at 100 g's for 1-2 o minutes to remove the spent l i q u o r s o l i d s . The supernatant was decanted and centrifuged at 2500 g's f o r 10 minutes to c o l l e c t the b a c t e r i a l c e l l s . A f t e r washing these separated c e l l s with d i s t i l l e d water, the wash water was removed by a further ce n t r i f u g i n g at 2500 g's for 10 minutes. This washing and c e n t r i f u g i n g procedure was done twice. Then the c e l l s were dispersed i n 10 times the o r i g i n a l sample volume of d i s t i l l e d water. For t e s t tube growth t e s t s no d i l u t i o n was used to measure the t u r b i d i t y . The t u r b i d i t y of the suspension was measured at 550 my i n a Bausch and Lomb spectronic 20 spectrophotometer, and c e l l suspension Concentration 14 was reported as t u r b i d i t y . See appendix 1. 2-6 Vitamin B 1 2 analysis Analysis f o r VB^ 2 m a v be c a r r i e d out i n various ways (79, 80). B i o l o g i c a l assays (81, 82, 83), m i c r o b i o l o g i c a l assays (84-90), isotope analysis (91, 92), paper chromatography (42), or spectrophotometric techniques (93, 94) may be employed. B i o l o g i c a l assays require f a c i l i t i e s f o r measuring r a t or chick growth, isotope analysis requires s p e c i a l i z e d equipment not presently available to us, and paper chromatography analysis requires c a l i b r a t i o n with hard to get pure cobamides. Thus we chose spectrophotometric analysis because of i t s s i m p l i c i t y and the a v a i l a -b i l i t y of equipment. However t h i s method i s not s p e c i f i c enough to d i s t i n g u i s h VBi2 from i t s various n o n - b i o l o g i c a l l y active analogs. Thus spectrophotometric measurements had to be checked by a m i c r o b i o l o g i c a l assay (85). In order to determine the VB 1 2 content of the c e l l s , the c e l l s were removed from the fermentation broth by centrifuging at 2500 g's for 15 minutes. A f t e r washing with d i s t i l l e d water the c e l l s were broken by heating or addition of acetone. In measurements of the V B j 2 content of the c e l l free broth or of the broth including the c e l l s , corrections had to be made f o r the e f f e c t s of spent l i q u o r components on the spectro-photometric measurements. The spectrophotometric technique was a modification of the method described by Rudk'ifi . and Taylor (93) and Fisher (94) . As previously noted there are several types of cobalamins (hydroxo, chloro, n i t r o , e t c . ) . These can r e a d i l y be converted to 15 cyanocobalamin by treatment with cyanide ions. In an a l k a l i n e s o l u t i o n containing an excess of cyanide ion cyanocobalamin takes on a second cyanide group to form a dicyanide cobalamin (purple coloured complex) which i s unstable, e x i s t i n g only i n an excess of cyanide ion. The col o r i m e t r i c assays of Rudkih and Taylor,and Fisher are based on the difference i n the v i s i b l e absorption spectra of cyanocobalamin and i t s dicyanide complex formed i n an excess of cyanide ion. Some of the components o f the s u l f i t e spent l i q u o r tend to i n t e r f e r e with t h i s a n a l y t i c a l procedure. V B 1 2 analysis according to Rudjcih- and Taylor i s as follows: "1. In a well v e n t i l a t e d hood s o l i d sodium cyanide i s added to a sample of the unknown containing approximately 200 micrograms of VB 1 2, a t a concentration of not less than 1 microgram per ml, so that the f i n a l concentration of cyanide i s 1%. The sample i s s t i r r e d to dissolve the sodium cyanide and the pH i s adjusted to 9.5 - 10.0 with 10% sodium hydroxide s o l u t i o n i f necessary. 2. This i s allowed to stand for 5 hours at room temperature for complete conversion of the V B i 2 to the dicyanide complex. 3. S o l i d sodium s u l f a t e (20% w./v.) i s added and dissolved. The pH i s further adjusted with sodium hydroxide to pH 11.0 to 11.5 and the aqueous s o l u t i o n i s extracted three times with one tenth volume of benzyl alcohol. 4. To the combined benzyl alcohol extracts one-half volume of chloroform i s added and the solvent phase i s extracted three times with one-tenth volume of water. The aqueous phase i s made up to 25 ml. 16 5. To a 10 ml aliquot of the water extract, 2 ml of a 10% sodium cyanide s o l u t i o n i s added. To another 10 ml a l i q u o t , 2 ml of a 12.5% potassium dihydrogen phosphate s o l u t i o n i s added to adjust the pH to 5 to 6. 6. The o p t i c a l density of each s o l u t i o n i s measured at 582 my. 7. The VB12 amount i s calculated from the d i f f e r e n c e , (AE)obsd, 1% i n the o p t i c a l d e n s i t i e s at 582 my. AE, 0 = 54 for c r y s t a l l i n e VBio. r 1cm J A^ 8. T o t a l V B 1 2 a c t i v i t y i n sample _ (AE)obsd. x 6/5 x 25 x 1.03 eq (1) 0.0054 x (cm c e l l length) where 1.03 i s an extraction f a c t o r . " Spectra for cyanocobalamin and i t s dicyanide complex are shown in F i g . 4. These are f o r pure VB 1 2 purchased from the B r i t i s h Drug Houses Ltd. Note that spectra i n t e r s e c t at 524,554 and 650 my. The spectra of s u l f i t e spent l i q u o r containing 200 yg V B 1 2 P e r sample i s shown i n F i g . 5. Also the absorption spectra of s u l f i t e spent l i q u o r are shown i n F i g . 6. Due to interference of the spent l i q u o r , the o p t i c a l density d i f f e r e n c e at 582 my, ^M^^ i n Fig* 5, cannot be used as (AE)obsd. to calculate V B i 2 a c t i v i t y . AE at 582 my due to the spent l i q u o r , AEs, can be calculated according to Beer's law by i n t e r p o l a t i o n of AE > AEj-j.^ and A E ^ ^ as these are due to spent l i q u o r only. In the case of Fig. 5 AE „. = 0.050, AE C C y l = 0.036, A E r o „ = 0.059, and AE, r r i = 0.016. b / 4 b o 4 o o Z 60U The o p t i c a l density d i f f e r e n c e at 582 my due to spent l i q u o r i s calculated to be 0.029. Thus (AE)obsd. = A E 5 g 2 - AE S = 0.059 - 0.029 = 0.030. From eq. (1) the V B i 2 a c t i v i t y i s calculated to be 173 yg, the actual value was 200 yg. 20 The m i c r o b i o l o g i c a l assay used Lactobacillus leichmanii, (ATCC >7830) as the t e s t organism. This i s a 16 to 24 hour turbidimetric procedure based on the U.S.P. XVI method (84). This i s done as follows: 1. To f i v e t e s t tubes (approx. 20 x 150 mm) add 1.0, 2.0, 3,0, 4.0 and 5.0 ml r e s p e c t i v e l y of the cyanocobalamin standard reference s o l u t i o n . This i s done i n t r i p l i c a t e . Now s u f f i c i e n t d i s t i l l e d water to make up each volume to 5.0 ml i s added. 2. To another series of tubes, the same amount, of the test s o l u t i o n of the material to be assayed i s added i n the same manner as outlined i n step 1, again i n t r i p l i c a t e . Then s u f f i c i e n t d i s t i l l e d water to make 5.0 ml i s added. 3. Another 3 t e s t tubes with 5.0 ml of d i s t i l l e d water are prepared as blanks. 4. To each tube 5.0 ml of basal medium stock s o l u t i o n (Bacto-Difco B 1 2;assay medium U.S.P. No. 0541-15) i s mixed. 5. A s e p t i c a l l y , one drop of inoculum i s added to each tube except one of the 3 blank tubes. Tube contents are mixed and incubated at 37°C for 16-24 hours. There should be no substantial increase i n t u r b i d i t y i n the tubes containing the highest l e v e l of standard during a 2 hour period. 6. The t u r b i d i t y of the cultures i s read i n a su i t a b l e photo-metric instrument at a s p e c i f i c wavelength between 540 and 660 my. In making readings, the contents of each tube should be thoroughly mixed. Afte r mixing tube contents are t r a n s f e r r e d to o p t i c a l glassware, and the tur-b i d i t y i s read. 7. Using the uninoculated blank, the meter of the : ^ instrument i s 21 set to read 100% transmittancy, then the transmittancy of the inoculated blank is read. If the inoculated blank tubes give a transmittancy of less than 65%, this indicates interference due to VB 1 2 activity in the basal medium stock solution, or the inoculum. 8. Using the inoculated blank and standard specimen, a standard response curve is prepared by plotting the % transmittancy reading for each level of standard cyanocabalamin solution used. A smooth curve should be drawn through the plotted points. One of standard curves which was made in this experiment is shown in Fig. 7. 9. Using this standard curve, by interpolation the amount of cyanocobalamin equivalent to the VBi 2 activity of each ml of the test sample under assay is determined. 2-7 Sulfite spent liquor preparation Calcium and ammonium base sulfite spent liquors were obtained from the pilot plant digesters of Columbia Cellulose Research and Develop-ment Division. The analysis of these liquors is presented in Table 3. Raw sulfite spent liquor contains much residual SO2 from the pulp cook. A large proportion of loosely bound S02 is believed to be attached to the wood sugars in the form of 2-hydroxy sulfonate as shown in Fig. 8. Some S02 may merely be dissolved in the spent liquor. As 200 ppm of S02 is sufficient to sterilize the liquor against bacteria (72), the S02 concentration must be reduced below this level. S02 removal was brought about by adjusting the pH of the liquor to about 5.5 with CaC03. The precipitate which resulted was removed by fi ltration. Finally, nutrients were added; these were yeast extract (1% by weight), cobalt chloride (5 ppm), Table 3 Typical Analysis of S u l f i t e Spent Liquor from Columbia Cellulose (Ca base) PH 1.6 Total s o l i d s 128 gr/1 Total sugars 37.4 gr/1 Fermentable sugars 64% of t o t a l sugars Lignosulfonate 79.3 gr/1 The Composition of a Spruce S u l f i t e Spent Liquor by Forss To t a l s o l i d s 12 - 16% of SSL Lignosulfonate 52% of t o t a l s o l i d s Monosaccharides 23% Poly and Oligo saccharides 6% Organic acids 7% Calcium 5% Sugar sulfonate 3% Others 4% " 25 and potassium dihydrogen orthophosphate (0.4% by weight). This solu-t i o n was then tested for i t s a b i l i t y to support b a c t e r i a l growth of P. f r e u d e n r e i c h i i , and i t i s r e f e r r e d to as "non-treated" medium. "Treated" medium was prepared by taking non-treated medium before the addition of nutrients and adjusting i t s pH to 12 with Ca(0H ) 2 « This caused further p r e c i p i t a t i o n which was assumed to be calciumligno-sulfonate (73). The p r e c i p i t a t e was f i l t e r e d o f f , the pH readjusted to between 6 and 7 with ^SO^, and nutrients were added as f o r the non-treated medium. Growth te s t s were then performed. When the pH of the l i q u o r was held at 12^more and more p r e c i p i t a t e was formed. Since the p r e c i p i t a t e contains a c e r t a i n amount of wood sugars, the sugar content o the treated media could be roughly adjusted at t h i s stage i n the t r e a t -ment by allowing more or less p r e c i p i t a t e to form. P r e c i p i t a t i o n could be accelerated by heating. Since the l i g n i n compounds are mainly res-ponsible for the colour of the s u l f i t e spent l i q u o r (74, 75), the treated medium was much clearer than the non-treated medium. Elements such as i r o n , zinc, and copper which are e s s e n t i a l f o r b a c t e r i a l growth were assumed to be present i n small quantities i n s u l f i t spent l i q u o r and were therefore not added. The addition of cobalt chloride to the media must be c a r e f u l l y c o n t r o l l e d since i t has been reported that a cobalt l e v e l above 50 ppm i n h i b i t s the synthesis of VB 1 2 (39, 76, 77, 78). 2-8 Synthetic media preparation For comparison with the s u l f i t e spent l i q u o r base media, the same amount of nutrient as i n a treated and non-treated media and 1% 26 glucose and a l i t t l e calcium carbonate (less than 0.05%) were added to d i s t i l l e d water and i s r e f e r r e d to as synthetic medium. This medium was also used to keep the culture. 2-9 Fermentation technique For the inoculum, P. f r e u d e n r e i c h i i from a synthetic medium containing 1% beef extract, 0.3% yeast extract, 1% peptone, 0.5% dextrose, 0.1% soluble starch, 0.05% cysteine hydrochloride, 0.5% sodium chloride, 0.3% sodium acetate, and 0.05% agar was transferred 24 hours a f t e r i t s inoculation, from tomato j u i c e agar to the treated or non-treated medium i n a test tube. A f t e r several transfers from t e s t tube to t e s t tube the b a c t e r i a changed i t s shape to cocci as described before and showed good growth i n treated media. 0.5 ml of culture was used to inoculate every 5 ml of fermentation;medium. 2-9-1 S t e r i l i z a t i o n S t e r i l i z a t i o n of media was ca r r i e d out i n an autoclave at 120°C. Depending on the volume of media, the s t e r i l i z a t i o n time was varied from 10 minutes to 45 minutes. 2-9-2 Test tube growth t e s t Test tube growth te s t s were c a r r i e d out using cotton plugged, stationary t e s t tubes. To measure the b a c t e r i a l growth i n the t e s t tubes, te s t tubes f o r the spectronic 20 were used and t u r b i d i t y was measured 48 hours to 120 hours a f t e r i n o c u l a t i o n . In these tests the media were not d i l u t e d before t u r b i d i t y readings were made. 27 2-9-3 Be l l c o glass fermentor The type of glass fermentors used i n t h i s experiments were Bellco glass fermentors (100 ml and 500 ihl). The fermentation temperature was 30 '+ 0.5°C. Ag i t a t i o n speed was 0.5 - 4 rps. Sampling was done using a s t e r i l i z e d pipet through a sample port which was plugged with cotton. The pH was not adjusted during these experiments since pH was not considered to be an important f a c t o r i f the i n i t i a l pH was between 5.5 and 7. A l l fermentations were done with treated calcium base spent liquo r media. 2- 9-4 7 I fermentation This was c a r r i e d out using a 7H fermentor (Bio-Kulture; Fer-mentation Design INC.). The diagram of t h i s fermentor i s shown i n F i g . 9.. The fermentation temperature was c o n t r o l l e d at 30°C, a g i t a t i o n speed was about 3 rps; the pH was not c o n t r o l l e d . As noted before i n section 1-3, only a g i t a t i o n was employed, aeration was not employed. Sampling was done using a s t e r i l i z e d pipet. The c e l l bodies were c o l l e c t e d with a Sharpies super centrifuge a f t e r fermentation. 3 Results 3- 1 Spectrophotometric analysis f or V B 1 2 The r e s u l t s of a check on the modified spectrophotometric technique f o r measuring V B 1 2 i n the presence of s u l f i t e spent l i q u o r are presented i n Table 4 and F i g . 10. Comparison of measured amounts of V B i 2 with the amounts added indicates reasonable agreement. 3-2 Comparison of spectrophotometric and m i c r o b i o l o g i c a l methods The spectrophotometric and m i c r o b i o l o g i c a l VB 1 2 assays are Table 4 Test of Accuracy of the Spectrophotometric Method for V B 1 2 Analysis Actual V B 1 2 content (yg) Measured V B 1 2 content (yg) 400 433 300 254 200 152 200 206 48 25 0 -6 169 154 169 161 0 -5 169 158 200 175 169 191 169 185 169 175 0 0 30 31 compared i n Table 5 and F i g . 11. Agreement can be said to be reasonable. 3-3 Test tube growth t e s t s . As shown i n Table 6 and 7 no b a c t e r i a l growth could be detected i n the non-treated spent l i q u o r medium. The only times that growth was observed i n the non-treated medium were when s u f f i c i e n t synthetic medium was added with the inoculum that the b a c t e r i a could grow on i t . In treated s u l f i t e spent l i q u o r medium good growth was observed as shown i n Table 7 and F i g . 12. Without yeast extract no growth was observed i n the calcium base l i q u o r . This of course indicates that some external source of nitrogen i s necessary. The fact that only s l i g h t growth was observed i n ammonia base l i q u o r i n the absence of yeast extract indicates the need f o r a nitrogen source other than ammonia. Reasonable growth was observed i n the ammonia base liquo r a f t e r the addition of yeast extract. 3-4 Bellco glass fermentor and 11 fermentor Results obtained from a number of fermentations i n Bellco glass fermentors (250 ml and 500 ml) with treated s u l f i t e spent l i q u o r medium are recorded i n Tables 8 to 10. F i g . 13 i s a graphical presentation of the r e s u l t s of one of these fermentations. Similar data f o r 7 l i t e r fermentations with both synthetic and treated calcium base spent l i q u o r media are provided i n Table 11 and Figs. 14 and 15. Table 5 Comparison of M i c r o b i o l o g i c a l and Spectrophotometric Assay VB 1 2 a c t i v i t y (pg/1) VB 1 2 a c t i v i t y (yg/1) Sample Spectrophotometric M i c r o b i o l o g i c a l assay assay SSL 210 220 small fermentor 380 530 (500 ml) 590 760 860 1100 830 960 Synthetic 320 350 7 £ fermentor 480 500 930 780 2050 1620 SSL -30 75 7'i fermentor 120 135 210 113 195 225 333 465 480 650 Table 6 Test Tube Growth Test i n Non-Treated Liquor 34 Base D i l u t i o n r a t i o pH at Start Yeast (%) Peptone (%) Growth Ca undiluted 5 1.0 0 No 5 0 1.0 No " 5 0 0 No 5 0.1 1.0 No 6 0.1 1.0 No 7 0.1 1.0 No 12.5 S 6 2 1.0 0 Yes 4 6 3 1.0 0 Yes 5 5 5 1.0 0 No 3 5 5 1.0 0 No 2 5 5 1.0 0 No undiluted 5 5 1.0 0 No NH3 undiluted 5 3 1.0 0 No I I 6 1 1.0 0 No 2 1.0 0 No 2 0 0 No 4 1.0 0 No 4 0 0 No undiluted 6 1 0 0 No 5 1.0 o No 3 1.0 0 No 2 1.0 0 No D i l u t i o n r a t i o with S Synthetic media + SSL media SSL media D i l u t i o n r a t i o D i s t i l l e d water + SSL media SSL media 35 Table 7 Test Tube Growth Test i n Treated Liquor Base D i l u t i o n r a t i o Sugar (g/1) pH Yeast (%) ' Growth Ca No d i l u t i o n 8.6 5.0 1.0 good No d i l u t i o n 15.1 5.0 1.0 good No d i l u t i o n 15.1 6.8 1.0 good 2 7.6 1.0 good 2 7.6 0 none 4 3.8 1.0 f a i r 4 3.8 0 none No d i l u t i o n 8.6 * 7.0 1.0 good No d i l u t i o n H 5.1 1.0 f a i r No d i l u t i o n 9.1 6.3 1.0 good 2 4.6 1.0 good No d i l u t i o n H 7.0 1.0 f a i r 2 H 1.0 p"oor NH3 No d i l u t i o n 14.7 6.1 1.0 poor 2 7.4 6.0 1.0 f a i r No d i l u t i o n 14.7 6.3 1.0 good No d i l u t i o n 14.7 6.3 0 poor 2 7.4 1.0 good 2 7.4 0 none 4 3.7 1.0 f a i r 4 3.7 0 none No d i l u t i o n 14.7 7.1 1.0 f a i r No d i l u t i o n H 7.3 1.0 f a i r No d i l u t i o n 6.6 1.0 f a i r 2 1.0 poor No d i l u t i o n H 6.3 1.0 f a i r 2 H 1.0 poor *H - Heated during p r e c i p i t a t i o n of l i g n i n . I 37 Table 8 Bellco Glass Fermentor Volume of fermentor (ml) Fermen-t a t i o n time (hr) Sugar (g/l) pH COD (mg/1) Acids (m mole/1) VB 1 2 (mg/D C e l l weight (g/D 100 0 118 2.14 0.99 6.1 4.9 53,000 49,000 44.7 0.7 500 0 118 3.7 0.9 5.8 5.0 71,000 53,500 1.03 2.04 100 0 168 2.5 0.4 5.5 4.9 60,700 52,000 53.1 500 0 168 7.5 3.2 5.7 5.0 68,000 52,000 100 0 120 7.5 5.9 5.75 4.6 61,120 56,700 0.6 1.47 500 0 120 9.5 7.7 5.92 4.7 69,840 63,800 0.8 1.65 500 No agitation 0 120 3.6 1.9 5.90 4.90 57,640 50,300 0.3 0.84 100 0 79 100 124 6.7 4.6 4.0 3.5 5.9 5.1 : 4.9 4.7 60,600 57,900 57,100 50,600 0.11 0.55 100 0 84 8.59 1.61 6.3 5.0 62,800 46,800 0.47 38 Table 9 Bellco Glass Fermentor Volume of Fermen- Sugar COD Acids VB 1 2 Turbi-fermentor t a t i o n (g/ D pH (mg/1) (m mole/1) (mg/1) d i t y (ml) time(hr) 500 0 6.7 5.75 59,700 80 4.5 5.00 57,900 33 0.075 100 4.0 4.80 57,100 0.46 124 3.6 4.80 50,600 41 0.51 148 2.7 4.80 46,300 0.48 172 0.4 4.75 44,000 53 0.81 0.81 C e l l weight 2.28 g / l 500 0 10.8 5.8 73,800 24 11.2 5.52 77,300 0.5 0.2 0.06 35 10.4 5.22 74,300 14.1 0.185 48 9.9 5.05 72,000 32.7 0.34 0.377 72 8.1 4.88 72,000 51.9 0.605 120 6.9 4.83 70,500 59.0 0.82 0.73 168 5.9 4.77 68,500 73.2 1.41 0.82 C e l l weight 2.83 g / l 100 0 12.1 6.4 73,100 12 12.1 6.2 3.5 0.03 24 11.3 5.9 7.3 0.07 48 10.1 5.1 34.2 0.39 72 7.6 4.75 68.3 0.61 120 6.2 4.7 81.3 0.70 168 5.12 4.7 58,000 87.5 1.05 0.73 C e l l weight 2.81 g / l 500 0 6.30 6.61 73,500 12 6.39 6.03 0.10 0.02 24 6.26 5.87 0.40 0.14 48 5.36 5.10 21.5 0.29 72 4.12 4.98 29.3 0.40 120 2.29 4.88 32.5 0.43 168 1.15 4.83 54,800 36.0 0.85 0.46 C e l l weight 2.19 g / l 39 Table 10 Bellco Glass Fermentor Volume of Fermen- Sugar COD Acids VB 1 2 Turbi-fermentor t a t i o n (g / D pH (mg/1) (m mole/1) (mg/1) d i t y (ml) time(hr) 500 0 10.9 5.87 79,400 0.021 12 9.80 5.41 62,500 2.4 0.072 24 9.01 5.23 58,100 8.5 0.21 0.140 48 6.78 5.11 54,600 23.6 0.38 0.247 72 4.73 5.00 46,600 32.4 0.59 0.383 96 3.81 4.85 46,800 34.9 0.86 0.515 120 2.85 4.83 46,200 36.3 0.83 0.521 C e l l weight 1.67 g / l 40 41 Table 11 11 Fermentor Fermentations Medium Time Sugar pH COD Acids VB 1 2 T u r b i d i t y (hr) (g/D (m mole/1) (mg/1) SSL 0 8.80 6.50 63,000 0 0 0.021 6 8.00 62,700 0 24 7.20 5,80 61,300 0.3 0 0.041 48 7.10 5.30 59,800 4.4 0.12 0.082 72 6.35 5.00 53,000 8.1 0.21 0.148 96 5.40 4.85 52,300 13.6 0.195 0.196 120 4.83 4.80 50,800 22.8 0.333 0.264 144 4.52 4.81 49,500 44.3 0.48 0.296 C e l l weight 1.05 g / l Synthetic 0 9.60 6.7 22,800 0 0 0.053 7 0.095 24 8.60 5.75 10.0 0.32 0.364 31 0.48 0.445 48 5.24 5.03 17,600 26.3 0.93 0.512 55 4.67 4.85 14,700 1.50 0.533 120 1.24 4.50 11,900 40.5 2.05 0.540 C e l l weight 2.37 g / l 43 Table 12 V o l a t i l e Acids Sugar used V o l a t i l e Acids Degree of conversion* (g/D (g/D (%) 1.15 3.04 264 2.10 3.71 176 6.98 6.12 88 5.15 2.52 49 6.30 3.73 59 4.90 5.13 104 8.04 2.68 34 8.36 2.84 34 4.28 3.10 ..72 Average mol weight i s calculated according to the r e s u l t that the r a t i o between propionic and ac e t i c acid i s roughly 2 : 1 . 45 3-4-1 COD Results ind i c a t e that f o r a l l the fermentations analyzed, the COD value was reduced by fermentation. However the COD of the media a f t e r fermentation i s s t i l l high i n d i c a t i n g that compounds other than fermentable sugars are responsible f o r most of the COD of the treated spent l i q u o r a f t e r fermentation. A COD vs. sugar content graph i s shown i n Fig. 16. 3-4-2 V o l a t i l e acids From Figs. 10, 12, and 13 one can see from the general shape of the curves of v o l a t i l e acids production and t u r b i d i t y vs. time that acids production seems to c l o s e l y follow c e l l production. 3-4-3 VB 1 2 Figs. 10, 12, and 13 show that V B 1 2 content of the media, including the c e l l s , increases with time. In one curve a f a l l i n g o f f was noted a f t e r 100 hours (Fig. 10). Much higher le v e l s of V B 1 2 con-centration were achieved i n the synthetic media. D i s t r i b u t i o n of V B 1 2 a c t i v i t y i n the c e l l s and i n the c e l l free media i s shown i n Table 13 for the 11 fermentations. 46 Table 13 VB12 A c t i v i t y i n 11 Fermentation (By L. leichmanii method) Medium Whole broth VB12 (mg/1) Medium liquor VB12 (mg/1) C e l l body VB 1 2 (mg/g dry c e l l ) C e l l growth (g / D Calculated t o t a l SSL 0.65 0.213 0.444 • 1.05 0.679 Synthetic 1.62 0.275 0.463 2.37 1.37 48 4. Discussion The spectrophotometric method requires less time than the microb i o l o g i c a l assay i f only a few samples are to be analyzed. However the m i c r o b i o l o g i c a l assay lends i t s e l f to the determination of the VB 1 2 content of a large number of samples. It has the further advantage of req u i r i n g a much smaller sample volume. I f the required equipment i s available the m i c r o b i o l o g i c a l assay i s to be preferred since t h i s method i s s p e c i f i c f o r b i o l o g i c a l l y active V B 1 2 . During the extraction of the V B ^ from the spent l i q u o r with benzyl alcohol i n the spectrophotometric assay, foaming of the spent liquo r i n t e r f e r e s , e s p e c i a l l y with non-treated l i q u o r . Sometimes t h i s foam d i d not disappear a f t e r 24 hours; these samples were discarded as the dicyanide complex of V B 1 2 i s not stable a f t e r 24 hours. This i n t e r -ference due to foaming at the l i q u i d - l i q u i d i n t e r f a c e may i n t e r f e r e with the extraction f a c t o r used by Rudkin and Taylor (93) i n c a l c u l a t i n g the V B i 2 content. Note that i n F i g . 10 the best l i n e through the measured r e s u l t s f a l l s somewhat below the expected l i n e . This foaming problem does not a r i s e i n the m i c r o b i o l o g i c a l assay nor f o r that matter was foaming a problem i n the larger scale fermentations. Although the m i c r o b i o l o g i c a l assay i s preferable i t involves a great deal of tedious p i p e t t i n g . The spectrophotometric assay i s reasonably easy to do and gives an acceptable r e s u l t as evidenced by Table 4. Chaiet e t . a l . (79) noted quite d i f f e r e n t r e s u l t s i n measuring the V B i 2 content of animal protein f a c t o r (APF) by each of these techniques) we encountered no such discrepancies. While there i s some scatter evident 49 i n F i g . 11, the microbiological assay does not give r e s u l t s c o n s i s t e n t l y higher than, or lower than the spectrophotometric assay r e s u l t s as was noted i n F i g . 11. In the untreated medium growth was not observed, hence we assume that as good growth occurs i n the treated medium, the lack of growth i n the untreated medium must have been due to the presence of to x i c agents (eg. SO2)which were removed i n the l i g n o s u l f o n i c acid p r e c i p i t a -t i o n stage. Growth i n ammonia base, treated media was not as good as i n calcium base, treated media. The reason f o r t h i s i s not known with c e r t a i n t y but we may speculate that a l i g n i n condensation reaction (75), va r i a t i o n s i n the polysaccharide-monosaccharide r a t i o , or va r i a t i o n s i n the s u l f u r compounds may be responsible. Batch fermentations usually follow a c e r t a i n sequence. F i r s t there i s a lag phase i n which the b a c t e r i a adapt themselves to the medium. C e l l mass increases but l i t t l e or no c e l l d i v i s i o n occurs. This i s followed by a log phase i n which the s p e c i f i c growth rate i s constant. Then a stationary phase i s observed i n which the c e l l s are a l i v e but not di v i d i n g due to the lack of some nutrient or the presence of some tox i c factor. F i n a l l y there i s a decline or death phase wherein the c e l l s d ie. This behaviour occurs when a single n u t r i t i o n a l f a c t o r i s l i m i t i n g . The growth, as measured by t u r b i d i t y of P.freudenreichii i n a synthetic media, which contains only a sin g l e sugar, follows t h i s pattern. This can be seen i n F i g . 14. Perhaps t h i s i s more c l e a r l y shown i n F i g . 17 where the volumetric rates are p l o t t e d against time. F i g . 17 was derived from 50 Fig. 14. Note that there i s only a single peak i n the t u r b i d i t y curve and i n the sugar comsumption curve. When more than one sugar i s available for the metabolic pro-cesses of the microorganism i t may use one p r e f e r e n t i a l l y . When the supply of t h i s sugar i s exhausted the organism adapts i t s e l f to using another. However t h i s may involve another lag period. This sort of behaviour can be seen i n Eigs. 12, 13, 15, 18, and 19. In the rate curves (Figs. 18 and 19) the sugar comsumption rate curves have two peaks as do the t u r b i d i t y curves. This may be due to the p r e f e r e n t i a l u t i l i z a t i o n of one type of sugar by the ba c t e r i a ; perhaps hexoses are used before pentoses. The long lag phase noted i n Fig. 15 may be due to the fact that the b a c t e r i a i n the inoculum were not properly adapted to the spent liquor. A new batch of s u l f i t e spent l i q u o r was used i n t h i s experiment. The volumetric basis fermentation rate curves (Bigs. 17, 18 and 19) show how the rates of production of c e l l s , V B i 2 and organic acid production vary with time. They also show the rate of substrate consump-t i o n . In the synthetic media the growth curve appears to follow the sugar consumption curve and the product curves (VBj 2 a n d organic acids) seem to follow the growth ( t u r b i d i t y ) curve. In the s u l f i t e spent liqu o r medium the picture i s not quite so c l e a r . The rate of production of acid and VB 1 2 follow the growth (tur-b i d i t y ) curve but the sugar consumption and growth curves do not show trends s i m i l a r to those observed with the synthetic media. As was men-tioned e a r l i e r , the b a c t e r i a have the choice of a number of sugars i n Shan, 54 the spent l i q u o r and the manner i n which they are consumed i s not shown by a o v e r a l l sugar analysis. R i l e y et.al.(64) noted the aeration was necessary to convert a precursor of V B 1 2 i n t o active VB^* Aso et. al.(33) suggested that s u f f i c i e n t aeration could be provided through the a i r l i q u i d i n t e r f a c e at the top of the fermentor i f a moderate amount of a g i t a t i o n was supplied. Our r e s u l t s would tend to confirm those of Aso. No aeration by bubbling was used. However there was opportunity for a i r to get i n t o the fermentor during f i l l i n g , during sampling, or through leakage. Each fermentation was agitated and b i o l o g i c a l l y active V B i 2 was produced. It should be noted that s t e r i l i z a t i o n was important and the time i n the s t e r i l i z e r should be c a r e f u l l y chosen. One 7 l i t r e fermen-t a t i o n was contaminated by a black mold which r a p i d l y took over the f e r -mentation from the b a c t e r i a . During s t e r i l i z a t i o n a large amount of greyish-white, sweet smelling, p r e c i p i t a t e was formed i n the s u l f i t e l i q u o r . This was not observed i n the synthetic media and was assumed to be some wood component. This p r e c i p i t a t e was f i l t e r e d o f f before the medium was inoculated. The COD of the medium a f t e r fermentation i s s t i l l high although somewhat lower than that measured before fermentation. (See Figs. 13, 14, and 15.) It should be noted that the COD values a f t e r fermentation were measured with the a c e t i c and propionic acid s t i l l present. In a commercial operation these would be removed and the COD reduced somewhat more. (.114, 115) With the synthetic medium (Fig. 14) the r e s i d u a l COD i s low and may be a t t r i b u t e d to the organic acids present. However t h i s fermentation appa-r e n t l y does nothing to the l i g n i n type compounds of the spent liquor. 55 Also i t must be remembered that these COD values were measured on treated liq u o r from which a good deal of pollutants had already been removed. With ce r t a i n wastes containing substances which are not consumed by b a c t e r i a , COD values may be higher than BOD values. However with wastes which contain only organic, b a c t e r i a l foods the COD value i s i n good agreement with the 20 day BOD value. Usually a 5 day BOD i s measured as a standard t e s t , t h i s value represents about 60% of the t o t a l oxygen demand. In s u l f i t e spent l i q u o r there are many chemicals such as tannins which are not r e a d i l y oxidizable by b a c t e r i a . Since the COD t e s t i s much easier and f a s t e r to perform than the BOD t e s t and because i t can be applied to a wider v a r i e t y of materials i t was chosen for t h i s work to ind i c a t e the change i n p o l l u t i o n p o t e n t i a l caused by the fermentation. The r a t i o of propionic acid to a c e t i c acid was measured and found to be 2.29 for the s u l f i t e l i q u o r medium and 2.05 for the synthetic medium. These r e s u l t s were measured on the 7 l i t r e fermentations. According to some workers (67), the t h e o r e t i c a l conversion of sugar to v o l a t i l e acids i s 77%. This i s based on the following equation. 3C 6H 1 20 6+ 4CH3CH2C00H + 2CH3COOH + 2C0 2 + 2H20 Following t h i s reasoning conversion r a t i o s of sugar to v o l a t i l e acids were calculated and are given i n Table 12. Some values are higher than 100% e s p e c i a l l y i n the fermentations with low i n i t i a l sugar content. This suggests that the above equation i s wrong or that the Sieber method does not c o r r e c t l y measure the sugar content of the medium before fermentation. 56 It may be unable to detect sugars i n a polymeric form: these could be hydrolyzed during fermentation to produce more monosaccharides which could then be fermented. In any case the equation given above does not apply to pentoses which are present, and which can be used by t h i s b a c t e r i a . Also,as the metabolic pathway f o r any organism i s rather complex i t would seem that such a simple stoichiometric equation i s a rather naive d e s c r i p t i o n of the true s i t u a t i o n . According to P f e i f e r et. a l . (63) the t o t a l V B 1 2 a c t i v i t y increases r a p i d l y f o r about 70-120 hours. A f t e r t h i s the V B i 2 a c t i v i t y i n the medium increases to some extent but the V B i 2 a c t i v i t y i n s i d e the c e l l s decreases. In t h i s work the d i s t r i b u t i o n of V B i 2 i n s i d e or outside of the c e l l was not measured as a function of time. Only a terminal r e s u l t was measured fcr the 7 l i t r e fermentations which i s given i n Table 13. To extract the V B i 2 from the medium would require an expensive l i q u i d -l i q u i d e x traction stage capable of t r e a t i n g large volumes of l i q u i d . However i f the major part of the V B i 2 i s inside the c e l l s , t h e c e l l s can be e a s i l y recovered by ce n t r i f u g a t i o n . Then the c e l l s could be broken and the V B i 2 recovered by a much smaller scale l i q u i d - l i q u i d e xtraction. The V B 1 2 i n the medium could be recycled to the fermentation to supply a growth f a c t o r for the organism. Since the goal of the project was to produce crude VB^ 2 s u i t a b l e f or animal feed i t would be desirable to have i t retained inside the c e l l s f o r reasons of economy of separation and also the c e l l bodies, i f not t o x i c , could be u t i l i z e d by the animal as a pro-t e i n source. 57 5. Conclusions The spent liqu o r from calcium and ammonium base s u l f i t e process pulp m i l l s can be fermented with Propionibacterium freuden-r e i c h i i , a f t e r a portion of the l i g n i n has been p r e c i p i t a t e d out by pretreatment. The products of the fermentation based on the spent l i q u o r from a one ton per day pulp m i l l , according to the r e s u l t s found i n t h i s study, would be: -2 0.6 to 1.5 x 10 pounds of t o t a l VB 1 2 (both i n c e l l bodies and medium) 15 to 20 pounds of a c e t i c a c i d 30 to 43 pounds of propionic acid Besides these marketable products there would r e s u l t a moderate reduction i n the COD value of the remaining spent li q u o r . The r a t i o of propionic acid to a c e t i c acid i n the product was shown to agree with the l i t e r a t u r e (67) value of two. However i n the case of the spent l i q u o r fermentation the s l i g h t increase of t h i s r a t i o to 2.3 may indi c a t e that a d i f f e r e n t metabolism i s occurring. The con-cl u s i o n that the fermentation mechanism i s d i f f e r e n t f or a synthetic medium containing a sin g l e monosaccharide and for s u l f i t e spent l i q u o r i s f o r t i f i e d by the shape and p o s i t i o n of the volume based rate curves f o r the fermentation. For rapid V B 1 2 assays, the spectrophotometric method modified f o r s u l f i t e spent l i q u o r gives good r e s u l t s . 6. Recommendations To reduce the oxygen demand of s u l f i t e spent l i q u o r and produce V B 1 2 and v o l a t i l e acids, the process shown i n F i g . 20 i s recommended. 58 59 Further work which should be undertaken on the s u l f i t e spent l i q u o r fermentation i s : (a) Continuous fermentation of 'treated' s u l f i t e spent l i q u o r . (b) Fermentation of the lignosulfonate p r e c i p i t a t e to give c e l l bodies or another marketable chemical product. (c) The timewise analysis of sugars i n the spent l i q u o r media. (d) Time study of the d i s t r i b u t i o n of V B 1 2 i n s i d e and outside the c e l l . (e) Attempt to ferment the non-treated s u l f i t e spent l i q u o r . Acknowledgement I wish to thank Dr. R. M. R. Branion and Dr. K. L. Pinder, under whose d i r e c t i o n t h i s i n v e s t i g a t i o n was conducted, f o r t h e i r guidance throughout t h i s study. I also wish to thank Dr. G. A. Strasdine of the Fisheries Research Board of Canada and Dr. J . J . Stock, Mrs. 0. Volkoff and Mrs. A. Schau i n Microbiology Department f o r t h e i r i n s t r u c t i o n i n m i c r o b i o l o g i c a l techniques. I wish also to thank Dr. L. R. Galloway, Columbia Cellulose for supplying the s u l f i t e spent l i q u o r and Miss J . Araki f o r typing. I am indebted to the University of B r i t i s h Columbia and the P a c i f i c Coast Branch of the technical section of Canadian Pulp and Paper Association f o r f i n a n c i a l support. 61 References 1. GEHM, H. W. , I n d u s t r i a l Waste Water C o n t r o l , Academic P r e s s , New York , 1965, p. 357 2. KHAZANOV, S . I. , A b s t . B u l l , I n s t , o f Paper Chem., 31 ; 8514 (1961) 3 . Washington F i s h e r i e s Research B u l l . , No. 5 , 264 (1960) 4. JONES, J . R. E . , F i s h and R i v e r P o l l u t i o n , B u t t e r w o r t h , London, 1964 5 . ECKENFELDER, W. W., and O'CONNOR, D. J . , B i o l o g i c a l Waste Treatment , Pergamon P r e s s , London, 1961 6. RYDHOLM, S . A . , P u l p i n g P r o c e s s , I n t e r s c i e n c e , New York , 1965 7. LIBBY, C. E . , Pu lp and Paper S c i . and Tech. v o l . 1 , Mcgraw H i l l , New York , 1962 8. CASEY, J . P . , Pu lp and Paper v o l . 1 , I n t e r s c i e n c e , New York , 19g2 9 . FORSS, K . , [Ph.D. T h e s i s ] , C e n t r a l l a b Ab. F i n n . Pu lp and Paper Research I n s t . , H e l s i n k i , F i n l a n d (1961) 10. GURNHAM, F . , I n d u s t r i a l Waste Water C o n t r o l , Academic P r e s s , New York (1965) 11 . YUKNA, A. D. and 0'ZOLIN'SH,A. P . , A b s t . B u l l , I n s t , o f Paper Chem., 35 ; 270 (1964) 12. ZELLSTOFF FABRIK WALDHOF, A b s t . B u l l , I n s t , o f Paper Chem., 3 5 ; 384 (1964) 13. McFARLANE, H. M . , Can. p a t . 696, 732 (1964) 14. McFARLANE, H. M . , Pu lp Paper Mag. Can. 6 3 ; No. 1 1 , T551-55 (1962) 15 . SHEVCHENKO, M. A . , and KAS'YANCHUK, R. S . , A b s t . B u l l , I n s t , o f Paper Chem., 35 ; 1282 (1965) 16. MARCHALL, A . , Can. p a t . , 642, 692 (1962) , U.S. p a t . , 2 , 9 5 2 , 507 (1960) 17. BUTLER, W. J . , P u l p . Paper Mag. C a n . , 5 0 ; No. 1 1 , 108 (1949) 18. SCHOLANDER, A . , T a p p i . 35 ; No. 1 , 1 (1952) 19. EDLING, G . , Paper Trade J . , 1 3 9 ; No. 2 1 , 28 (1955) 20. TOPPEL, V. 0 . , Das P a p i e r , 15; 81 (1961) 62 21. BRAUNS, F. E., The Chemistry of Lignin, Academic Press, New York 1952 22. HORIO, M., et a l , Kogyo-Kagaku Gairon, Chu-kan, Maruzen, Tokyo (1960) 23. PERRY, H. J . , et a l , Proc. 14th Ind. Waste Conf., Pardue Univ. Eng. B u l l ; 44, No. 5, 200 (1960) 24. EL'PINER, I. E., Ultrasound, Consultants Bureau, New York, 1964 25. CARLSON, C , et a l , Svensk Papperstid, 61; 162 (1958) 26. ENEBO, L., et a l , Svensk Papperstid, 61; 162 (1958) 27. DILLEN, S., Svensk Papperstid, 64; 283,545,819 (1961) 28. JENSEN, 0., Parasitenk I I , 4; 217,265,325 (1898) 29. VAN NIEL, C. B., Tech, Hoogeschool, Delft (1928) 30. LEVITON, A. and HARGROVE, R. E., Ind. Eng. Chem., 44; 2651 (1952) 31. PERLMAN, D., Adv. i n Appl, Microbiol, 1; 87 (1959) 32. HODGE, H. M., HANSON, C. T.and ALLGEIER, R. J . , Ind. Eng. Chem., 44; 132 (1952) 33. ASO, K., ARITA, M., et a l , Nippon Nogei-Kagaku Kaishi, 28; 111 (1954) 34. WAYMAN, M. et a l , U.S. Pat., 3,067,107 (1962) 35. MARTIN, M. and WAYMAN, M., Can. J . Microbiol, 7; 342 (1961) 36. YAMADA, K., Hokkaido Forest Products Research Inst. Report, C.F.ABIPC, 34; No. 6, 3958 (1954) 37. GRAF, G. and MARTIN, M., Columbia C e l l , Lab. Status Report PR-LSR-356 (1958) 38. PERLMAN, D., Adv. i n Appl, Microbiol, 7; 103 (1965) 39. KOJIMA, H. and MATSUKI, M., Tohoku J . Agr. Research, 7; 175 (1956) 40. RICKES, E. L., BRINKS, N. G. et a l , Science, 108; 134 (1948) 41. RICKES, E. L., BRINKS, N. G. et a l , Science, 108; 634 (1948) 42. PERLMAN, D. and BARRETT, J. M., J . B a c t e r i d . , 78; 171 (1959) 43. TAMAO, Y., KATO, T. et a l , J. Vitamin (Japan), 28; 579 (1963) 44. EL-HAGARAWY, I. S., et a l , Dairy S c i . , 40; 579 (1957) 63 45. WERKMAN, C. H. and BRAUN, R. W., J . Bacterid. , 26; 393 (1933) 46. FLAVIN, M. and OCHOA, S., J . Biol. Chem., 229; 965 (1957) 47. FLAVIN, M. and CASTRO-MENDOZA, H . , J . Biol. Chem., 229; 981 (1957) 48. BECK, W. S., FLAVIN, M. et a l , J . Biol. Chem., 229; 987 (1957) 49. BECK, W. S. and OCHOA, S., J . Biol. Chem., 230; 931 (1958) 50. PHARES, E. F. and DELWICHE, E. A . , J . Bacterid. , 71; 609 (1956) 51. SMITH, R. M. and MONTY, K. J . , Biochem. Biophys. Research Commum., 1; 105 (1959) 52. SWICK, R. W., Proc. Am. Chem. Soc, Atlantic city, 70C (1959) 53. DELWICHE, E. A. and CARSON, S. F . , J . Bacterid. , 65; 318 (1953) 54. BERGEY'S MANUAL OF DETERMINATIVE BACTERIOLOGY, Williams and Wilkins, Baltimore, 1957 55. STADTMAN, E. and OVERATH, P., Biochem. Biophys. Research Commun, 2; 1 (1960) 56. KATO, T. and SHIMIZU, S., J . Vitamin (Japan), 26; 473 (1962) 57. SHIMIZU, S. and KATO, T . , J . Vitamin (Japan), 26; 470 (1962) 58. KATO, T . , TAMAO, Y. et a l , J . Vitamin (Japan), 28; 579 (1963) 59. HODGKIN, D. C , PICKWORTH, J . et a l , Nature, 176; 325 (1955) 60. SMITH, E. L . , Nutr. Abstr. and Revs., 20; 795 (1950-51) 61. WOOD, H. G. et a l , Archiv. Biochim. Biophys., 49; 249 (1954) 62. GARIBALDI, J . A. and IJICHI, K., Ind. Eng. Chem., 45; 838 (1953) 63. PFEIFER, v. F . , VOJNORIECH, C. and HEGER, E. N. , Ind. Eng. Chem., 46; 843 (1954) 64. RILEY, P. B. et al , Soc. Chem. Ind. (London) Monograph, 12; 127 (1961) 65. SUDARSKY, J . M. and FISHER, R. A . , U.S. pat. 2,816,856 (1957) 66. SIEBER, R., Die Chemiech-Technischen Untersuchungs-Methoden der Zellstoff und Papier Industrie, Springer Publishing, 1951, p. 344 67. BERNHAUER, K., Columbia Cell . Status Report, PR-LSR-356 (1958) p. 5 64 68. PARTANSKY, A. M. and BENSON, H. K., The Chemistry o f L i g n i n , Academic Press, New York, 1951, p. 172 69. Standard Methods f o r the Examination of Water and Waste Water, 11th ed., Am. P u b l i c Health Assoc., New York, 1960, p. 399 70. SCHMIDT, U., Papier, 15; 79 (1961) 71. ADLER, E., Svensk P a p p e r s t i d , 50; 261 (1947) 72. WEBB, F. C , Biochemical Engineering, Van Nostrand, London, p. 350 (1964) 73. HOWARD, G. C , Ind. Eng. Chem., 22; 1184 (1930) 74. HAGGLUND, E., Chemistry o f Wood, Academic Press, New York, 1951, Chapt. 3 75. IVANCIC, A. and RYDH0LM, S. A., Svensk P a p p e r s t i d , 62; 554 (1959) 76. PETTY, M. A., U.S. pat. 2,515,135 (1950) 77. PERLMAN, D. and SEMAR, J . B., Abstr. 138th Meeting Am. Chem. Soc. P10A (1960) 78. PERLMAN, D., Adv. i n Appl, M i c r o b i o l . , 7; 103 (1965) 79. CHAIET, L. and MILLER, T., J . Agr. Food Chem., 2; 785 (1954) 80. WILLIAMS, W. L. and STIFFEY, A. V., J . Agr. Food Chem., 4; 364 (1956) 81. NICHOL, C. A., DIETRICH, L. S. et a l , Proc. Soc. Exper. B i o l , and Med., 70; 40 (1949) 82. OTT, W. H., RICKES, E. L. et a l , P o u l t r y S c i , 30; 86 (1951) 83. BOSSHARDT, D. K., PAUL, W. J . et a l , J . Nutr., 37; 21 (1949) 84. KRIEGER, C. H., Assoc. of O f f i c i a l Agr. Chem., 38; No. 3, 711 (1955) 85. PERLMAN, D., YACOWITZ, H. and WEISER, H. H., P o u l t r y S c i . , 33; 1043 (1954) 86. FORD, J . E., B r i t . J . Nutr., 7; 299 (1953) 87. COATES, M. E. and FORD, J . E., Biochem. Soc. Symposia (Cambr. E n g l . ) , 13; 36 (1955) 88. COATES, M. E., and KON, S. K., Vitamin B 1 2 and I n t r i n s i c F a c t o r , Ferdinand Enke V e r l a g , S t u t t g a r t , 1957, p. 96 89. KON, S. K., Biochem. Soc. Synposia, 13; 17 (1955) 65 90. DAVIS, B. D. and MINGIOLI, E. S., J . B a c t e r i d . , 60; 17 (1950) 91. BACHER, F. A. and BOLEY, A. E., A n a l y t i c a l Chem., 26; 1147 (1954) 92. MONIER, D., GHALIOUNGHI, Y. and SABA, R., Anal. Chem. Acta., 28; 30 (1963) 93. RUDKIN, G. 0. and TAYLOR, R. J . , Anal. Chem., 24; 1155 (1952) 94. FISHER, R. A., J . Agr. Food Chem., 1; 951 (1953) 95. HODGKIN, D. C , PICKWORTH, J . et a l , Nature, 176; 325 (1955) 96. PFIFFNER, J . J . and CALKINS, D. G., Div. of B i o l . Chem. 120th Meeting Anal. Chem. Soc. Abstr. o f Papers, p. 22C (1951) 97. PORTER, J . W. G. and DOLLAR, A. M., I n t e r . Congr. Biochem. 4th Congr. Vienna Abstr. Papers, p. 96 (1958) 98. FORD, J . E., et a l , Biochem. J . 50; 4 (1951) 99. DiMARCO, A., B o l l . Soc. I t a l . B i o l . Sper., 33; 1513 (1957) 100. PAWELKIEWITZ, J . , Acto. Biochem. Polon., 6; 431 (1959) 101. BARCHIELLI, R. and B0RETTI, G., Biochem. Biophys. Acta., 25; 452 (1957) 102. FORD, J . E. and PORTER, J . W. G., B r i t . J . Nutr., 7; 326 (1953) 103. HINZ, C. F., B a c t e r i a l Process, Soc. Am. B a c t e r i a l , p. 26 (1957) 104. PAGONA, J . F. and GREENSPAN, G., U.S. pat. 2,695,864 (1954) 105. DULANEY, E. L. and WILLIAMS, P. L., Mycologia, 45; 345 (1953) 106. HALL, H. H. and BENEDICT, R. G., Appl. M i c r o b i o l . , 1; 124 (1953) 107. HESTER, A. and WARD, G., Ind. Eng. Chem., 46; 238 (1954) 108. NELSON, H. A., et a l , Abst. 118th Meeting Am. Chem. Soc. p. 16A (1950) 109. LEWIS, U. J . and IJICHI, K., U.S. Dept. Agr. B u l l . AIC 254 (1949) 110. WOOD, T. R. and HENDLIN, D., U.S. pat. 2,595,499 (1952) 111. KON, S. K. and PAWELKIEWICZ, J . , Proceed of 4th I n t e r . Congr. Biochem., Vienna, v o l . 9, p. 115 (1958) 112. HEINRICH, H. C , Proceed o f 4th I n t e r . Congr. Biochem., Vienna, v o l . 9, p. 150 (1958) 66 113. PETERSON, B. H., HALL, B., and BIRD, 0. D., J . B a c t e r i o l . , 71; 91 (1956) 114. MOORE, W. A., et a l , Anal. Chem. S o c , 21; 953 (1949) 115. MOORE, W. A., et a l , Anal. Chem. S o c , 23; 1297 (1951) 67 Appendix 1-1 Sugar a n a l y s i s T o t a l sugar (reducing substance) i n s u l f i t e spent l i q u o r was measured by Rudolf Sieber method (Swedish Standard) (66) . The procedure i s as f o l l o w s . 1. S u l f i t e spent l i q u o r i s n e u t r a l i z e d with CaC0 3 and d i l u t e d 10 times w i t h water. 2. 10 ml copper s u l f a t e s o l u t i o n (69.3 g CuSO4.5H.2O per l i t e r ) , 10 ml se i g n e t t e s a l t s o l u t i o n (350 g C^H^Og KNa.4H20 and 100 g NaOH per l i t e r ) , and 20 ml d i s t i l l e d water are heated t o b o i l i n g i n a f l a s k w i t h i n 3-4 minutes. As soon as the sample has come to the b o i l , a 10 ml of n e u t r a l i z e d and d i l u t e d sample i s added from a p i p e t t e w i t h i n 15 seconds. Within 60 seconds the sample s o l u t i o n should resume b o i l i n g . E x a c t l y 3 minutes a f t e r a d d i t i o n o f sample, the b o i l i n g i s discontinued and the f l a s k i s a b r u p t l y cooled to about 20°C under the c o l d water tap. 3. While s w i r l i n g , 10 ml o f potassium i o d i d e s o l u t i o n (300 g KI per l i t e r ) and then 10 ml of s u l f u r i c a c i d (250 g H2SO4 i n l i t e r ) are added t o the f l a s k . 4. This s o l u t i o n i s t i t r a t e d with 0.1N sodium t h i o s u l f a t e s o l u t i o n and the consumption of 0.1N sodium t h i o s u l f a t e , expressed i n ml, i s recorded as "a". 5. D i s t i l l e d water i s s u b s t i t u t e d i n place o f the s u l f i t e spent l i q u o r and the above procedure i s repeated. The consumption o f 0.1N sodium t h i o s u l f a t e s o l u t i o n at t h i s t i t r a t i o n i s recorded as "b". 6. Copper s u l f a t e s o l u t i o n i s replaced by 10 ml o f d i s t i l l e d water. In t h i s case t i t r a t i o n i s c a r r i e d out with 0.1N io d i n e s o l u t i o n . The consumption of i o d i n e s o l u t i o n i s recorded as "c". ) 68 7. The t o t a l consumption "T" of 0.1N sodium t h i o s u l f a t e which corresponds t o the sugar content i n 10 ml d i l u t e d or 1 ml o r i g i n a l s u l f i t e spent l i q u o r i s c a l c u l a t e d as f o l l o w s . T = [b - a - c] ml 8. The conversion o f T t o reducing sugar i s done with reference to the t a b l e 77 i n "Die Chemisch-Technischen Untersuchungs-Methoden der Z e l l s t o f f und Papier' I n d u s t r i e " , p. 344 (66). Appendix 1-2 V o l a t i l e a c i d s a n a l y s i s To determine the v o l a t i l e acids 10 ml of sample were a c i d i f i e d with 0.5 - 1 ml of 85% phosphoric a c i d . This mixture was heated t o 130 -140°C i n an o i l bath to d i s t i l l o f f the v o l a t i l e a c i d s . A f t e r 8-10 ml of d i s t i l l a t e were c o l l e c t e d , 10 ml of water was added to the content of the d i s t i l l i n g f l a s k and the d i s t i l l a t i o n was repeated t i l l 20-25 ml of d i s t i l l a t e was c o l l e c t e d . The t o t a l volume o f d i s t i l l a t e was t i t r a t e d with 0.100N NaOH. C a l c u l a t e d on the b a s i s o f 1.000N NaOH and of 1000 ml of sample, the volume o f c a u s t i c consumed gave the number o f m i l l i m o l e s of v o l a t i l e a c i d s . Appendix 1-3 Ratio of p r o p i o n i c a c i d t o a c e t i c a c i d (37) "Using the v o l a t i l i t y w i t h steam and based on the presence of p r o p i o n i c and a c e t i c a c i d o n l y , the r a t i o o f a c e t i c and p r o p i o n i c a c i d was c a l c u l a t e d . 50 ml a c i d i f i e d sample was b o i l e d under r e f l u x f o r 10 minutes t o e x p e l l carbon d i o x i d e ; r e p l a c i n g the r e f l u x w i t h a condenser, the sample was s t e a m - d i s t i l l e d u n t i l 400 ml d i s t i l l a t e had been c o l l e c t e d ; the sum of v o l a t i l e acids was determined by t i t r a t i n g a p o r t i o n of the d i s t i l l a t e ( a ) ; another 200 ml was subjected to h a l f d i s t i l l a t i o n ; 100 ml 69 of d i s t i l l a t e was c o l l e c t e d and t i t r a t e d with 0.100N NaOH (b). I f a = t o t a l a c i d i t y i n 200 ml b = a c i d i t y i n 100 ml h a l f d i s t i l l a t e then a = HAC + HPr b = 0.366 HAc + 0.585 HPr where HAC and HPr are expressed i n terms of ml c a u s t i c . An unsuccessful attempt was made to determine t h i s r a t i o by a gas chromatographic technique. This would probably be a more con-venient and accurate method i f proper columns were a v a i l a b l e . " Appendix 1-4 L i g n i n measurement The determination of l i g n i n was c a r r i e d out as f o l l o w s (68). 10 cc of the s u l f i t e spent l i q u o r was b o i l e d i n a small beaker f o r 10-15 minutes to e x p e l l the free s u l f u r d i o x i d e . The evaporated water i s repl a c e d and 10 cc of a 5% s o l u t i o n o f 2-naphthylamine hydro-c h l o r i d e i n 3N h y d r o c h l o r i c a c i d was added while s t i r r i n g . This mixture was heated on a steam bath f o r 1 hour. The p r e c i p i t a t e formed began to coalesce and soon changed to a s o f t gummy consistency. On c o o l i n g i t became hard and b r i t t l e . I t was f i l t e r e d on a weighed Gouch f i l t e r , completely a i r d r i e d over n i g h t , and f i n a l l y heated t o 100°C. The amount of l i g n o s u l f o n a t e was c a l c u l a t e d according to the equation g.Lignosulfonate = g.NLS x 0.793 x 1.22 where NLS i s the p r e c i p i t a t e d naphthylamine l i g r o s u l f o n a t e , 0.793 i s a f a c t o r f o r l i g n o s u l f o n a t e i n NLS, and 1.22 i s the f a c t o r to c o r r e c t f o r the l i g n i n that i s not p r e c i p i t a t e d by the naphthylamine. 70 Appendix 1-5 T u r b i d i t y Transmittancy (T) i s the r a t i o o f the r a d i a n t energy t r a n s -mitted by the sample (P) to the energy i n c i d e n t upon the sample (Po). Both r a d i a n t energies must be obtained at the same wavelength, w i t h the same s p e c t r a l s l i t width. T = P/Po Transmittancy i s u s u a l l y given i n percent. Since i t i s seldom p o s s i b l e to measure these r a d i a n t energies d i r e c t l y because of the presence of a sample c e l l , i t i s customary to consider the transmittancy of the sample as the r a t i o o f the l i g h t t r a n s m i t t e d by the c e l l and the sample t o the l i g h t t r a n s m i t t e d by some a r b i t r a r y standard. In other words, the sample i s compared to a standard. The transmittancy of t h i s standard i s defined as 100 percent. Absorbancy, t u r b i d i t y , or o p t i c a l d e n s i t y are the negative logarithm t o the base 10 of the transmittancy: - log T = log 1/T = log Po/P where T i s expressed as a decimal f r a c t i o n , not i n percent. T u r b i d i t y i s a standard t o measure the c e l l growth, and o p t i c a l d e n s i t y i s used f o r the absorbancy o f the s o l u t i o n . Appendix 2 COD This i s ASTM D 1252-58T method which i s c a r r i e d out as f o l l o w s . 1. A 50 ml sample or an a l i q u o t d i l u t e d to 50 ml with d i s t i l l e d water i s plac e d i n a round-bottom f l a s k and 25 ml of 0.25N standard pota-ssium dichromate s o l u t i o n i s added. The f l a s k i s attached to a F r i e d r i c h s condenser and the mixture i s r e f l u x e d f o r 2 hours. I t i s then cooled 71 and the condenser i s washed down with about 25 ml d i s t i l l e d water. 2. The contents are t r a n s f e r r e d to a 500 ml c o n i c a l f l a s k , washing out the r e f l u x f l a s k 4-5 times w i t h d i s t i l l e d water. The mixture i s d i l u t e d to about 350 ml and a f t e r c o o l i n g to room temperature the excess dichromate i s t i t r a t e d w i t h 0.25N standard ferrous ammonium s u l f a t e , using f e r r o i n i n d i c a t o r . 3. A blank c o n s i s t i n g o f 50 ml d i s t i l l e d water i n s t e a d of the sample, together w i t h the reagents, i s r e f l u x e d i n the same manner. 4. Oxygen demand i s c a l c u l a t e d according to the f o l l o w i n g equation. where C 0 D = ( a - b ) c x 8,000 6 ml sample a = ml Fe(NIT\) 2 ( S O 4 ) 2 used f o r blank b = ml Fe ( N H i t ) 2 (50^)2 used f o r sample c = n o r m a l i t y of Fe ( N H i J 2 (SOtJ 2 The r a t e and extent of oxygen d e p l e t i o n i s customarily evalu-ated by the biochemical oxygen demand, BOD, t e s t . This i s not a d i r e c t measure of organic content, but i s a measure of the c a p a c i t y to consume oxygen using a s u i t a b l e microorganism. The BOD t e s t has the disadvantage that i t r e q u i r e s 5 days f o r completion. COD t e s t does not d u p l i c a t e the BOD t e s t . I t may or may not have a c o n s i s t e n t r a t i o to BOD, however there i s no f u l l y acceptable s u b s t i t u t e f o r the 5-day BOD t e s t . Both COD and BOD t e s t s measure organic substances, but some of organic compounds are r e s i s t a n t t o each of the two t e s t s , and not i n the same manner. KOKI Eigo dewa umaku iia r a w a s e n a i bubun mo aru node aratamete kokode sukosi nobetai. Ryo su p e r v i s o r no kakumen n i wataru enjo wa i u n i oyobazu d o s h i t u no Mr. R. G. Orr ga SSL no n i o i n i yoku taenuki mata r e g i s t r a t i o n sonota no enjo o oshimazu ataete kureta koto nimo o i n i kansha suru. Mr. Sugano, Mr. Shima, Mr. Uchinami no benkyo, torampu, majan to tahomen n i wataru shido nimo o i n i kansha suru. Chika no Dr. Kitamura, Dr. Watanabe nimo t a t a ou tokoro ga o i . Mr. Kubo, Mr. Koga no shido n i y o r i gorufu, t e n i s u , tozan to o i n i tanoshinda. Saigoni kono k i k a i o ataete kureta U.B.C. Chemical Engineering Department n i kansha suru. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0059117/manifest

Comment

Related Items